Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, FIN-90014 Oulu, Finland.
Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
J Mol Biol. 2018 Sep 14;430(18 Pt B):3081-3092. doi: 10.1016/j.jmb.2018.06.048. Epub 2018 Jul 5.
Histone lysine demethylases (KDMs) are 2-oxoglutarate-dependent dioxygenases (2-OGDDs) that regulate gene expression by altering chromatin structure. Their dysregulation has been associated with many cancers. We set out to study the catalytic and inhibitory properties of human KDM4A, KDM4B, KDM5B, KDM6A and KDM6B, aiming in particular to reveal which of these enzymes are targeted by cancer-associated 2-oxoglutarate (2-OG) analogues. We used affinity-purified insect cell-produced enzymes and synthetic peptides with trimethylated lysines as substrates for the in vitro enzyme activity assays. In addition, we treated breast cancer cell lines with cell-permeable forms of 2-OG analogues and studied their effects on the global histone methylation state. Our data show that KDMs have substrate specificity. Among the enzymes studied, KDM5B had the highest affinity for the peptide substrate but the lowest affinity for the 2-OG and the Fe cosubstrate/cofactors. R-2-hydroxyglutarate (R-2HG) was the most efficient inhibitor of KDM6A, KDM4A and KDM4B, followed by S-2HG. This finding was supported by accumulations of the histone H3K9me3 and H3K27me3 marks in cells treated with the cell-permeable forms of these compounds. KDM5B was especially resistant to inhibition by R-2HG, while citrate was the most efficient inhibitor of KDM6B. We conclude that KDM catalytic activity is susceptible to inhibition by tumorigenic 2-OG analogues and suggest that the inhibition of KDMs is involved in the disease mechanism of cancers in which these compounds accumulate, such as the isocitrate dehydrogenase mutations.
组蛋白赖氨酸去甲基酶（KDMs）是依赖 2-氧戊二酸（2-OG）的双加氧酶（2-OGDDs），通过改变染色质结构来调节基因表达。它们的失调与许多癌症有关。我们着手研究人类 KDM4A、KDM4B、KDM5B、KDM6A 和 KDM6B 的催化和抑制特性，特别是旨在揭示这些酶中哪些是癌症相关的 2-氧戊二酸（2-OG）类似物的靶标。我们使用亲和纯化的昆虫细胞产生的酶和具有三甲基化赖氨酸的合成肽作为体外酶活性测定的底物。此外，我们用可渗透细胞的 2-OG 类似物处理乳腺癌细胞系，并研究它们对全局组蛋白甲基化状态的影响。我们的数据表明 KDMs 具有底物特异性。在所研究的酶中，KDM5B 对肽底物具有最高的亲和力，但对 2-OG 和 Fe 辅因子/辅助因子的亲和力最低。R-2-羟基戊二酸（R-2HG）是 KDM6A、KDM4A 和 KDM4B 的最有效抑制剂，其次是 S-2HG。这一发现得到了细胞内组蛋白 H3K9me3 和 H3K27me3 标记物积累的支持，这些细胞用这些化合物的可渗透细胞形式处理。KDM5B 对 R-2HG 的抑制特别具有抗性，而柠檬酸是 KDM6B 的最有效抑制剂。我们得出结论，KDM 催化活性易受致癌性 2-OG 类似物的抑制，并表明 KDM 的抑制参与了这些化合物积累的癌症的疾病机制，例如异柠檬酸脱氢酶突变。
